Epothilone derivatives

ABSTRACT

The present invention relates to C4-demethyl-epothilones or C4-bisnor-epothilones of Formula (I), their pharmaceutical use, pharmaceutical composition containing the same and methods for their preparation.

The present invention relates to C4-demethyl-epothilones orC4-bisnor-epothilones and their pharmaceutical use, pharmaceuticalcomposition containing the same and methods for their preparation.

Despite the widespread use of Taxol® and Taxotere® in the treatment ofmany different tumor types, the impact of taxanes on patient survivalhas been modest, and the over-whelming majority of metastatic solidtumors remain incurable. Taxane treatment is associated with a number ofsignificant side-effects, and the effectiveness of taxanes can beseverely limited by the rapid development of drug resistance mechanisms.In view of these limitations as well as the side-effects commonlyobserved with standard combination therapies, there is a clear need forthe identification of novel cytotoxic anti-cancer agents exhibiting animproved overall profile including spectrum of anti-tumor activity,efficacy against multi-drug resistant tumors, safety and tolerability.

The microtubule-stabilizing effect of the epothilones was firstdescribed by Bollag et al., Cancer Research 55, 1995, 2325-33. Asuitable treatment schedule for the treatment of different types oftumors, especially tumors which are refractory to the treatment by otherchemotherapeutics, in particular TAXOL™, using an epothilone, inparticular epothilone A or B, is described in WO 99/43320. D. Su, A.Balog et al. discussed in Angew. Chem. Int. Ed. Engl. 1997, 36, pages2093 to 2096, the structure-activity relationship of the class of theepothilones. On pages 2094 of said publication, they inter aliaconcluded that a modification of the structure of the natural compoundsat the carbon atoms indicated as C1 to C8 results in a major loss ofcytotoxicity and of loss of activity in the tubulin/microtubule system.Surprisingly, it has now been found that the C4-(demethyl orbisnor)-epothilones of formula I have beneficial pharmacologicalproperties and can be used for the treatment of proliferative diseases.

Hence, the present invention relates to C4-(demethyl orbisnor)-epothilones of formula I

wherein A represents O or NR₇,

R₁ is hydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkanoyl in free or protected form, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino, orlower acyl amino,

R₂ is unsubstituted or substituted heteroaryl having at least onenitrogen atom,

R₃ represents hydrogen or lower alkyl, preferably methyl,

R₅ and R₆ are hydrogen, and

R₇ is hydrogen or lower alkyl,

Z is O or a bond,

under the proviso that

when R₂ is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkylwhich is unsubstituted or substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, and

when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ represents lower alkylwhich is substituted by hydroxy, lower acyloxy, lower alkoxy, halogen,amino, lower alkyl amino, di-lower alkyl amino or lower acyl amino,

and to the salts thereof.

The general terms used herein before and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise Indicated:

Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

Any asymmetric carbon atoms may be present in the (R)—, (S)— or(R,S)-configuration, preferably in the (R)— or (S)-configuration. Thecompounds may thus be present as mixtures of isomers or as pure Isomers,preferably as enantiomer-pure diastereomers.

The formula I represents two stereoisomers. The present inventionrelates to both such stereoisomers represented by formula Ia and Ib,

wherein the symbols and radicals have the meanings as provided for acompound of formula I above, preferably to a compound of formula Ia.

The prefix “lower” denotes a radical having up to and including amaximum of 7, especially up to and including a maximum of 4 carbonatoms, the radicals in question being either linear or branched withsingle or multiple branching.

“Halogen” is fluorine, chlorine, bromine or iodine.

“Alkyl” is preferably lower alkyl.

“Lower alkyl” is linear or branched; e.g. It is butyl, such as n-butyl,sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl,ethyl or methyl. Preferably lower alkyl is methyl, ethyl, n-propyl,isopropyl or tert-butyl.

“Alkoxy” is preferably lower alkoxy, e.g. methoxy, ethoxy, isopropoxy ortert-butoxy.

“Acyl” is preferably lower acyl, e.g. acetyl.

“Lower alkanol” is preferably methanol, ethanol, 1-propanol, 2-propanol,1-butanol or 2-butanol.

“Lower alkane” is in particular pentane, hexane or heptane.

“Heteroaryl having at least one nitrogen atom” represents a mono- orbicyclic group comprising at least one, two or three ring nitrogen atomsand zero or one oxygen atom and zero or one sulfur atom, which group isunsaturated in the ring binding the heteroaryl radical to the rest ofthe molecule in formula I and is preferably a group, where the bindingring preferably has 5 to 12, more preferably 5 or 6 ring atoms; andwhich may be unsubstituted or substituted by one or more, especially oneor two, substituents, preferably selected from halogen, alkoxy,alkylthio, hydroxy, alkanoyl or, most preferably, alkyl. Preferably themono- or bicyclic heteroaryl group is selected from 2H-pyrrolyl,pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, purinyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4H-quinolizinyl,isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalyl,quinazolinyl, quinnolinyl, pteridinyl, 3H-indolyl, indolyl, isoindolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, benzo[b]thiazolyl,triazolyl, tetrazolyl, benzo[b]oxazolyl and benzo[d]pyrazolyl.

In the presence of negatively charged radicals, such as carboxy orsulfo, salts may also be formed with bases, e.g. metal or ammoniumsalts, such as alkali metal or alkaline earth metal salts, for examplesodium, potassium, magnesium or calcium salts, or ammonium salts withammonia or suitable organic amines, such as tertiary monoamines, forexample triethylamine or tri(2-hydroxyethyl)amine, or heterocyclicbases, for example N-ethyl-piperidine or N,N′-dimethylpiperazine.

When a basic group and an acid group are present in the same molecule, acompound of formula I may also form internal salts.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

In view of the close relationship between the novel compounds in freeform and those in the form of their salts, including those salts thatcan be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the freecompounds hereinbefore and hereinafter is to be understood as referringalso to the corresponding salts, as appropriate and expedient.

The compounds of formula I have valuable pharmacological properties, asdescribed hereinbefore and hereinafter.

The efficacy of the compounds of formula I as inhibitors of microtubuledepolymerisation may be proved as follows:

Stock solutions of the test compounds (10 mM) are prepared in DMSO andstored at −20° C. Microtubule protein (i.e. tubulin plusmicrotubule-associated proteins) is extracted from pigs' brain by twocycles of temperature-dependent depolymerisation/polymerisation, asknown (see Weingarten et al., Biochemistry 1974; 13: 5529-37). Workingstock solutions of porcine microtubule protein are then stored at −70°C. The degree of test-compound-induced polymerisation of porcinemicrotubule protein is determined basically as already known (see Lin etal., Cancer Chem. Pharm. 1996; 38:136-140). To summarise, a workingaliquot of porcine microtubule protein is rapidly thawed and thendiluted to 2× final desired concentration in ice-cold 2× MEM buffer (200ml MES, 2 mM EGTA, 2 mM MgCl₂, pH 6.7) [MES=2-morpholinoethanesulphonicacid, EGTA=ethylene glycol-bis-2(2-aminoethyl)-tetraacetic acid]. Drugor vehicle (DMSO, final concentration 5%) is diluted in water at roomtemperature to 2× the final desired concentration in a 0.5 ml eppendorftube, and then put on ice. Following addition of an equal volume (50 μl)of microtubule protein (2× final desired concentration in 2× MEMbuffer), the polymerization reaction is started by transferring theincubation mixtures to a room-temperature water bath for 5 min. Thereaction mixtures are then placed in an Eppendorf microcentrifuge (model5415 C) and incubated for an additional 15 min at room temperature. Thesamples are then centrifuged for 15 min at 14,000 rpm at roomtemperature. As an indirect measure for microtubule proteinpolymerization, the protein concentration of the supernatant (containingthe remainder of non-polymerized, soluble microtubule protein) isdetermined by the Lowry method (DC Assay Kit, Bio-Rad Laboratories,Hercules, Calif.) and the optical density of the colour reaction ismeasured at 750 nm with a SpectraMax 340 photometer (Molecular Devices,Sunnydale, Calif.). The reduction in optical density by test drug iscompared to that induced by 25 μM epothilone B (positive control, 100%polymerization). Vehicle-treated samples serve as negative control (0%polymerization). Polymerization activity of test drug is expressed inpercentage relative to the positive control (100% polymerization).

The efficacy against tumour cells may be demonstrated in the followingway:

Stock solutions of the test compounds (10 mM) are prepared in DMSO andstored at −20° C. Human KB-31 and (multidrug-resistant, P-gp170overexpressing) KB-8511 epidermoid carcinoma cells originate from Dr. M.Baker, Roswell Park Memorial institute (Buffalo, N.Y., USA)(description: see also Akiyama et al., Somat. Cell. Mol. Genetics 11,117-126 (1985) and Fojo A., et al., Cancer Res. 45, 3002-3007 (1985)-KB-31 and KB-8511 are both derivatives of the KB cell line (ATCC). KB31 cells may be cultivated in monolayers using RPMI-1640 medium (Amimed,BioConcept, Allschwil, Switzerland) with 10% foetal calf serum (Amimed,BioConcept, Allschwil, Switzerland), L-glutamine (Amimed, BioConcept,Allschwil, Switzerland), penicillin (50 units/ml) and streptomycin (50μg/ml (Amimed, BioConcept, Allschwil, Switzerland). KB-8511 is a variantderived from the KB-31 cell line, which was obtained using colchichinetreatment cycles, and has an approximately 40 times relative resistanceto colchichine compared with KB-31 cells (Akiyama et al., Somat. Cell.Mol. Genetics 11, 117-126 (1985) and Fojo A., et al., Cancer Res. 45,3002-3007 (1985)). The cells are incubated at 37° C. in an incubatorwith 5% v/v CO₂ and at 80% relative humidity with RPMI-1640 mediumcomplemented as described above. The cells are seeded in a quantity of1.5×10³ cells/well in 96-well microtitre plates, and incubated overnight. Serial dilutions of the test compounds in culture medium areadded on day 1. The plates are then incubated for a further 4 days,after which the cells are fixed with 3.3% v/v glutaraldehyde, washedwith water and dyed with 0.05% w/v methylene blue. After washing, thedye is eluted with 3% HCl and the optical density measured at 665 nmwith a SpectraMax 340 (Molecular Devices, Sunnyvale, Calif.). IC50values are determined by adaptation of mathematical curves, using theSoftProprogramme (Version 2.0 or later; Molecular Devices, Sunnyvale,Calif.) and using the formula [(OD treated)−(OD start)]/[(ODcontrol)−(OD start)]×100. The IC50 is defined as the concentration of atest compound at the end of the incubation period, which led to 50%inhibition of the net increase in cell mass compared to controlcultures. Compounds of formula I thus preferably show an IC50 in therange of 0.15 and 15 nM, preferably between 0.25 and 5 nM.

The in vivo efficacy may be demonstrated as follows: The models used arexeno-transplants of tumours, such as KB-31 or KB-8511 epidermoidtumours, in mice. The anti-tumour efficacy of the test compounds may bemeasured in female BLB/c nu/nu mice for example against thecorresponding subcutaneously transplanted cell line. To this end, tumourfragments of about 25 mg are implanted into the left side of each of themice (for example 6 animals per dose). The test compound is administeredfor example on day 11 after transplantation in different dosages (forexample 0.1; 0.5; 1; 5 and 10 mg/kg), if desired repeating theadministration, if required several times, after between two days andtwo weeks. The volumes of the tumours are determined for example afterabout 2 to 4 weeks (e.g. two weeks after the start of treatment). Thetumour volumes are calculated by measuring the tumour diameter along twovertically arranged axes and according to published methods (see Evanset al., Brit. J. Cancer 45, 466-8 (1982)). The anti-tumour efficacy isdetermined as the mean increase in tumour volume of the treated animalsdivided by the mean increase in tumour volume of the untreated animals(controls) and, after multiplication by 100, is expressed as T/C%.Tumour regression (given in %) is calculated as the smallest mean tumourvolume (Vt) in relation to the mean tumour volume at the start oftreatment (Vo) according to the formula% regression=[1−(Vt/Vo)]×100.

In this case also, other cell lines can be used, for example those namedabove in the demonstration of efficacy against tumour cells.

On the basis of their efficacy as inhibitors of tubulin depolymerizationthe C4-desmethyl epothilone of the formula I are effective against anumber of proliferative diseases, such as solid tumor diseases, liquidtumor disases (like leukemia) or psoriasis.

The term “solid tumor disease” especially means breast cancer, cancer ofthe colon and generally the GI tract including gastric cancer, hepatoma;lung cancer, in particular small-cell lung cancer and non-small-celllung cancer, renal cancer, mesothelioma, glioma, squamous cell carcinomaof the skin, head and neck cancer, genitourinary cancer, e.g. cervical,uterine, ovarian, testicles, prostate or bladder cancer; Hodgkin'sdisease, carcinoid syndrome or Kaposi's sarcoma. In a preferredembodiment of the invention, the solid tumor disease to be treated isselected from breast cancer, colorectal cancer, ovarian cancer, renalcancer, lung cancer, especially non-small-cell lung cancer, and glioma.The C4-desmethyl epothilone of the formula I disclosed herein are alsosuitable to prevent the metastatic spread of tumors and the growth ordevelopment of micrometastases, in particular due to theirant-angiogenic activity.

A C4-desmethyl epothilone of formula I can be administered alone or incombination with one or more other therapeutic agents, possiblecombination therapy taking the form of fixed combinations or theadministration of a compound of the invention and one or more othertherapeutic agents being staggered or given independently of oneanother, or the combined administration of fixed combinations and one ormore other therapeutic agents. A C4-desmethyl epothilone of formula Ican be administered especially for tumor therapy in combination withchemotherapy, radiotherapy, immunotherapy, surgical intervention, or acombination of these. Long-term therapy is equally possible as isadjuvant therapy in the context of other treatment strategies, asdescribed above. Other possible treatments are therapy to maintain apatient's status after tumor regression, or even chemopreventivetherapy, for example in patients at risk.

Therapeutic agents for possible combination are especially one or moreantiproliferative, cytostatic or cytotoxic compounds, for example achemotherapeutic agent or several agents selected from the group whichincludes, but is not limited to, an inhibitor of polyamine biosynthesis,an inhibitor of a protein kinase, especially of a serine/threonineprotein kinase, such as protein kinase C, or of a tyrosine proteinkinase, such as the EGF receptor tyrosine kinase, erg. PKI166, the VEGFreceptor tyrosine kinase, e.g. PTK787, or the PDGF receptor tyrosinekinase, e.g. STI571, a cytokine, a negative growth regulator, such asTGF-β or IFN-β, an aromatase inhibitor, e.g. letrozole or anastrozole,an inhibitor of the interaction of an SH2 domain with a phosphorylatedprotein, antiestrogens, topolsomerase I inhibitors, such as irinotecan,topoisomerase II inhibitors, other microtubule active agents, e.g.paclitaxel, or (+)-discodermolide, alkylating agents, antineoplasticantimetabolites, such as gemcitabine or capecitabine, platin compounds,such as carboplatin or cisplatin, anti-anglogenic compounds, gonadorelinagonists, anti-androgens, bisphosphonates, e.g. AREDIA® or ZOMETA®, andtrastuzumab. The structure of the active agents identified by code nos.,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications). The corresponding contentthereof is hereby incorporated by reference.

In general, the invention relates also to the use of a C4-desmethylepothilone of formula I or a salt thereof for the stabilization of themicrotubule cell skeleton, either In vitro or In vivo.

With the groups of preferred C4-desmethyl epothilone of formula I andsalts thereof mentioned hereinafter, definitions of substituents fromthe general definitions mentioned herein before may reasonably be used,for example, to replace more general definitions with more specificdefinitions or especially with definitions characterized as beingpreferred.

In particular, the invention relates to a C4-desmethyl-epothilone offormula Ia or Ib

wherein A represents O or NR₇,

R₁ is hydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkanoyl in free or protected form, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino, orlower acyl amino,

R₂ is unsubstituted or substituted heteroaryl having at least onenitrogen atom,

R₃ represents hydrogen or lower alkyl, preferably lower alkyl

R₅ and R₆ are hydrogen, and

R₇ is hydrogen or lower alkyl,

Z is O or a bond,

under the proviso that when R₂ is 2-methyl-thiazolyl and Z is O, R₁represents lower alkyl which is unsubstituted or substituted by hydroxy,lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino, di-loweralkyl amino or lower acyl amino, and when R₂ is 2-methyl-thiazolyl and Zis a bond, R₁ represents lower alkyl which is substituted by hydroxy,lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino, di-loweralkyl amino or lower acyl amino,

and to the salts thereof.

Especially, the invention relates to C4-(demethyl or bisnor)-epothilonesof formula I or of formula Ia or Ib, wherein

A represents O or NR₇,

R₁ is hydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkanoyl in free or protected form, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino, orlower acyl amino,

R₂ is thiazolyl, oxazolyl, pyridyl, benzothiazolyl, benzoxazolyl orbenzoimidazolyl, which in each case is substituted or unsubstituted,

R₃ represents hydrogen or tower alkyl, preferably lower alkyl

R₅ and R₆ are hydrogen, and

R₇ is hydrogen or lower alkyl,

Z is O or a bond,

under the proviso that

when R₂ is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkylwhich is unsubstituted or substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, and

when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ represents lower alkylwhich is substituted by hydroxy, lower acyloxy, lower alkoxy, halogen,amino, lower alkyl amino, di-lower alkyl amino or lower acyl amino,

and the salts thereof.

Preferred are C4-(demethyl or bisnor)-epothilones of formula I or offormula Ia or Ib, wherein

A represents O or NR₇,

R₁ is hydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino,di-lower alkyl amino or lower acyl amino,

R₂ is thiazolyl, oxazolyl, pyridyl, benzothiazolyl, which in each caseis substituted or unsubstituted,

R₃ represents hydrogen or lower alkyl, preferably lower alkyl

R₅ and R₆ are hydrogen, and

R₇ is hydrogen or lower alkyl,

Z is O or a bond,

under the proviso that

when R₂ is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkylwhich is unsubstituted or substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, and

when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ represents lower alkylwhich is substituted by hydroxy, lower acyloxy, lower alkoxy, halogen,amino, lower alkyl amino, di-lower alkyl amino or lower acyl amino,

and the salts thereof.

More preferred are C4-(demethyl or bisnor)-epothilones of formula I orof formula Ia or Ib, wherein

A represents O,

R₁ is hydrogen or lower alkyl,

R₂ is 2-methyl-thiazolyl, 2-ethyl-thiazolyl, 2-methylthio-thiazolyl,2-aminomethyl-thiazolyl, 2-dimethylamino-thiazolyl,2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl, 3-methyl-pyridinyl,2-methyl-benzothiazolyl,

R₃ represents hydrogen lower alkyl, preferably lower alkyl,

R₅ and R₆ are hydrogen, and

Z is O or a bond,

under the proviso that when R₂ is 2-methyl-thiazolyl, Z is O and R₁represents lower alkyl, and the salts thereof.

Even more preferred are C4-(demethyl or bisnor)-epothilones of formula Ior of formula Ia or Ib, wherein

A represents O,

R₁ is hydrogen or lower alkyl,

R₂ is 2-methyl-thiazolyl, 2-ethyl-thiazolyl, 2-methylthio-thiazolyl,2-aminomethyl-thiazolyl, 2-dimethylamino-thiazolyl,2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl, 3-methyl-pyridinyl,2-methyl-benzothiazolyl,

R₃ represents methyl,

R₅ and R₆ are hydrogen, and

Z is O or a bond,

under the proviso that when R₂ is 2-methyl-thiazolyl, Z is O and R₁represents lower alkyl, and the salts thereof.

Furthermore, the present invention relates to the use of aC4-desmethyl-epothilone of formula I or of formula Ia or Ib or apharmaceutically acceptable salt thereof for the treatment of a tumourdisease and for the preparation of a pharmaceutical product for thetreatment of a tumour disease.

Additionally, the present invention provides a method for the treatmentof warm-blooded animals, including humans, in which an therapeuticallyeffective amount of a C4-desmethyl-epothilone of the formula I or offormula Ia or Ib or a pharmaceutically acceptable salt of such acompound is administered to a warm-blooded animal suffering from atumour disease.

Epothilones of formula I, wherein A represents O or NR₇, R₁ is hydrogenor lower alkyl which is unsubstituted or substituted by hydroxy, loweracyloxy, lower alkoxy, halogen, amino, lower alkyl amino, di-lower alkylamino, lower acyl amino, R₂ is unsubstituted or substituted heteroaryl,R₇ is hydrogen or lower alkyl, R₅ and R₆ are hydrogen and Z is O or abond can be prepared, e.g., by a process wherein an aldehyde of formulaII,

wherein R₁, R₂ and Z have the meanings as provided above for a compoundof formula I and R₄ is a protecting group, is reacted in a first stepwith an ethylketone of formula III,

wherein R₅ is H or a protecting group different or identical to R₄ andR₃ has the meaning as provided above for a compound of formula I, toprovide the aldol of formula IV,

wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, R₄ a protecting group, R₅ is H or a protectinggroup different or identical to R₄ and R₆ is hydrogen,

which aldol of formula IV is reacted in a second step with a reagentcapable to introduce a protecting group which is different or identicalto R₄ furnishing a carboxylic acid of formula IV, wherein R₁, R₂, R₃ andZ have the meanings as provided above for a compound of formula I, R₄ aprotecting group and R₅ is H or R₅ and R₆ are protecting groupsdifferent or identical to R₄,

which carboxylic acid of formula IV is reacted in a third step with areagent capable to remove the protecting group R₄ under conditions whichdo not result in the removal of the protecting groups R₅ and R₆providing a carboxylic acid of formula IV, wherein R₁, R₂, R₃ and Z havethe meanings as provided above for a compound of formula I, R₄ ishydrogen and R₅ is H or R₅ and R₆ are protecting groups,

which carboxylic acid of formula IV in a fourth step is subject of amacrolactonisation reaction providing the epothilone of formula I,wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, A is O and R₅ is H or R₅ and R₆ are protectinggroups,

which epothilone of formula I is reacted in a fifth step with a reagentcapable to remove the protecting groups R₅ (if existing) and R₆furnishing an epothilone of formula I, wherein R₁, R₂, R₃, R₅, R₆ and Zhave the meanings as provided above for a compound of formula I and A isO,

which epothilone of formula I is, optionally, further transformed intoan epothilone of formula I wherein R₁, R₂, R₃, R₅, R₆ and Z have themeanings as provided above for a compound of formula I and A is NR₇,wherein R₇ is hydrogen or lower alkyl.

Epothilones of formula Ia or Ib, wherein A represents O or NR₇, R₁ ishydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino,di-lower alkyl amino, lower acyl amino, R₂ is unsubstituted orsubstituted heteroaryl, R₇ is hydrogen or lower alkyl, R₅ and R₆ arehydrogen and Z is O or a bond can be prepared, e.g., by a processwherein an aldehyde of formula II,

wherein R₁, R₂ and Z have the meanings as provided above for a compoundof formula I and R₄ is a protecting group, is reacted in a first stepwith an ethylketone of formula IIIa or IIIb, respectively,

wherein R₆ is H or a protecting group different or identical to R₄ andR₃ has the meaning as provided above for a compound of formula I, toprovide the aldol of formula IVa or IVb, respectively,

wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, R₄ a protecting group, R₅ is H or a protectinggroup different or identical to R₄ and R₆ is hydrogen,

which aldol of formula IVa or IVb is reacted in a second step with areagent capable to introduce a protecting group which is different oridentical to R₄ furnishing a carboxylic acid of formula IVa or IVb,wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, R₄ a protecting group and R₅ is H or R₅ and R₆are protecting groups different or identical to R₄,

which carboxylic acid of formula IVa or IVb is reacted in a third stepwith a reagent capable to remove the protecting group R₄ underconditions which do not result in the removal of the protecting groupsR₅ and R₆ providing a carboxylic acid of formula IVa or IVb, wherein R₁,R₂, R₃ and Z have the meanings as provided above for a compound offormula I, R₄ is hydrogen and R₅ is H or R₅ and R₆ are protectinggroups,

which carboxylic acid of formula IVa or IVb in a fourth step is subjectof a macrolactonisation reaction providing the epothilone of formula Iaor Ib, wherein R₁, R₂, R₃ and Z have the meanings as provided above fora compound of formula I, A is O and R₅ is H or R₅ and R₆ are protectinggroups,

which epothilone of formula Ia or Ib is reacted in a fifth step with areagent capable to remove the protecting groups R₅ and R₆ furnishing anepothilone of formula Ia or Ib, wherein R₁, R₂, R₃, R₅, R₆ and Z havethe meanings as provided above for a compound of formula I and A is O,

which epothilone of formula Ia or Ib is, optionally, further transformedinto an epothilone of formula Ia or Ib wherein R₁, R₂, R₃, R₅, R₆ and Zhave the meanings as provided above for a compound of formula I and A isNR₇, wherein R₇ is hydrogen or lower alkyl.

Protecting Groups

If one or more other functional groups, for example carboxy, hydroxy,amino, or mercapto, are or need to be protected in a compound mentionedherein, because they should not take part in the reaction, these aresuch groups as are usually used in the synthesis of peptide compounds,and also of cephalosporins and penicillins, as well as nucleic acidderivatives and sugars.

The protecting groups may already be present in precursors and shouldprotect the functional groups concerned against unwanted secondaryreactions, such as acylations, etherifications, esterifications,oxidations, solvolysis, and similar reactions. It is a characteristic ofprotecting groups that they lend themselves readily, i.e. withoutundesired secondary reactions, to removal, typically by solvolysis,reduction, photolysis or also by enzyme activity, for example underconditions analogous to physiological conditions, and that they are notpresent in the end-products. The specialist knows, or can easilyestablish, which protecting groups are suitable with the reactionsmentioned hereinabove and hereinafter.

The protection of such functional groups by such protecting groups, theprotecting groups themselves, and their removal reactions are describedfor example in standard reference works, such as J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork 1973, in T. W. Greene, “Protective Groups in Organic Synthesis”,Wiley, N.Y. 1981, in “The Peptides”; Volume 3 (editors: E. Gross and J.Meienhofer), Academic Press, London and New York 1981, in “Methoden derorganischen Chemie” (Methods of organic chemistry), Houben Weyl, 4thedition, Volume 15/l, Georg Thieme Veriag, Stuttgart 1974, in H.-D.Jakubke and H. Jescheit, “Aminosäuren, Peptide, Proteine” (Amino acids,peptides, proteins), Veriag Chemie, Weinheim, Deerfield Beach, and Basel1982, and in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharideund Derivate” (Chemistry of carbohydrates: monosaccharides andderivatives), Georg Thieme Verlag, Stuttgart 1974.

R₅ can be a protecting group which is different or identical to R₄. Incase both protecting groups are identical, such groups have to representprotection groups which can be splitt off from the compound of formula Isequentielly, i.e. reaction conditions must exist for such groupsallowing to replace R₄ by hydrogen or another protecting group underwhich the protecting group R₅ remains in the compound of formula I.

The transformation of epothilone B to the corresponding lactam isdisclosed in Scheme 21 (page 31, 32) and Example 3 of WO 99/02514 (pages48-50). The transformation of a compound of formula I which is differentfrom epothilone B into the corresponding lactam can be accomplishedanalogously. Corresponding epothilone derivatives of formula I whereinR₄ is lower alkyl can be prepared by methods known in the art such as areductive alkylation reaction starting from the epothilone derivativewherein R₄ is hydrogen.

Additional Process Steps

In the additional process steps, carried out as desired, functionalgroups of the starting compounds which should not take part in thereaction may be present in unprotected form or may be protected forexample by one or more of the protecting groups mentioned hereinaboveunder “protecting groups”. The protecting groups are then wholly orpartly removed according to one of the methods described there.

Salts of a compound of formula I with a salt-forming group may beprepared in a manner known per se. Acid addition salts of compounds offormula I may thus be obtained by treatment with an acid or with asuitable anion exchange reagent.

Salts can usually be converted to free compounds, e.g. by treating withsuitable basic agents, for example with alkali metal carbonates, alkalimetal hydrogencarbonates, or alkali metal hydroxides, typicallypotassium carbonate or sodium hydroxide.

Stereoisomeric mixtures, e.g. mixtures of diastereomers, can beseparated into their corresponding isomers in a manner known per se bymeans of suitable separation methods. Diastereomeric mixtures forexample may be separated into their individual diastereomers by means offractionated crystallization, chromatography, solvent distribution, andsimilar procedures. This separation may take place either at the levelof a starting compound or in a compound of formula I itself. Enantiomersmay be separated through the formation of diastereomeric salts, forexample by salt formation with an enantiomer-pure chiral acid, or bymeans of chromatography, for example by HPLC, using chromatographicsubstrates with chiral ligands.

It should be emphasized that reactions analogous to the conversionsmentioned in this chapter may also take place at the level ofappropriate intermediates.

General Process Conditions

All process steps described here can be carried out under known reactionconditions, preferably under those specifically mentioned, in theabsence of or usually in the presence of solvents or diluents,preferably such as are inert to the reagents used and able to dissolvethese, in the absence or presence of catalysts, condensing agents orneutralizing agents, for example ion exchangers, typically cationexchangers, for example in the H⁺ form, depending on the type ofreaction and/or reactants at reduced, normal, or elevated temperature,for example in the range from −100° C. to about 190° C., preferably fromabout −80° C. to about 150° C., for example at −80 to −60° C., at roomtemperature, at −20 to 40° C. or at the boiling point of the solventused, under atmospheric pressure or in a closed vessel, whereappropriate under pressure, and/or in an inert atmosphere, for exampleunder argon or nitrogen.

Salts may be present in all starting compounds and transients, if thesecontain salt-forming groups. Salts may also be present during thereaction of such compounds, provided the reaction is not therebydisturbed.

At all reaction stages, isomeric mixtures that occur can be separatedinto their individual isomers, e.g. diastereomers or enantiomers, orinto any mixtures of isomers, e.g. racemates or diastereomeric mixtures,typically as described under “Additional process steps”.

The solvents from which those can be selected which are suitable for thereaction in question include for example water, esters, typically loweralkyl-lower alkanoates, e.g. diethyl acetate, ethers, typicallyaliphatic ethers, e.g. diethylether, or cyclic ethers, e.g.tetrahydrofuran, liquid aromatic hydrocarbons, typically benzene ortoluene, alcohols, typically methanol, ethanol or 1- or 2-propanol,nitrites, typically acetonitrile, halogenated hydrocarbons, typicallydichloromethane, acid amides, typically dimethylformamide, bases,typically heterocyclic nitrogen bases, e.g. pyridine, carboxylic acids,typically lower alkanecarboxylic acids, e.g. acetic acid, carboxylicacid anhydrides, typically lower alkane acid anhydrides, e.g. aceticanhydride, cyclic, linear, or branched hydrocarbons, typicallycyclohexane, hexane, or isopentane, or mixtures of these solvents, e.g.aqueous solutions, unless otherwise stated in the description of theprocess. Such solvent mixtures may also be used in processing, forexample through chromatography or distribution.

The invention relates also to those forms of the process in which onestarts from a compound obtainable at any stage as a transient andcarries out the missing steps, or breaks off the process at any stage,or forms a starting material under the reaction conditions, or uses saidstarting material in the form of a reactive derivative or salt, orproduces a compound obtainable by means of the process according to theinvention and processes the said compound in situ. In the preferredembodiment, one starts from those starting materials which lead to thecompounds described hereinabove as preferred, particularly as especiallypreferred, primarily preferred, and/or preferred above all.

In the preferred embodiment, a compound of formula I is preparedaccording to or in analogy to the processes and process steps defined inthe Examples.

The compounds of formula I, including their salts, are also obtainablein the form of hydrates, or their crystals can include for example thesolvent used for crystallization (present as solvates).

Pharmaceutical Preparations, Methods, and Uses

The present invention relates also to pharmaceutical compositions thatcomprise a compound of formula I as active ingredient and that can beused especially in the treatment of the diseases mentioned herein.Compositions for enteral administration, such as nasal, buccal, rectalor, especially, oral administration, and for parenteral administration,such as intravenous, intramuscular or subcutaneous administration, towarm-blooded animals, especially humans, are especially preferred. Thecompositions comprise the active ingredient alone or, preferably,together with a pharmaceutically acceptable carrier. The dosage of theactive ingredient depends upon the disease to be treated and upon thespecies, its age, weight, and individual condition, the individualpharmacokinetic data, and the mode of administration.

The invention relates also to pharmaceutical compositions for use in amethod for the prophylactic or especially therapeutic management of thehuman or animal body, to a process for the preparation thereof(especially in the form of compositions for the treatment of tumors) andto a method of treating tumor diseases, especially those mentionedhereinabove.

Pharmaceutical preparations contain from about 0.000001% to 95% of theactive ingredient, whereby single-dose forms of administrationpreferably have from approximately 0.00001% to 90% and multiple-doseforms of administration preferably have from approximately 0.0001to 0.5%in the case of preparations for parenteral administration or 1% to 20%active ingredient in the case of preparations for enteraladministration. Unit dose forms are, for example, coated and uncoatedtablets, ampoules, vials, suppositories or capsules. Further dosageforms are, for example, ointments, creams, pastes, foams, tinctures,lipsticks, drops, sprays, dispersions, etc. Dose unit forms, such ascoated tablets, tablets or capsules, contain about 0.0025 g to about 0.1g of the active ingredient.

The pharmaceutical preparations of the present invention are prepared ina manner known per se, for example by means of conventional mixing,granulating, coating, dissolving or lyophilising processes.

Preference is given to the use of solutions of the active ingredient,and also suspensions or dispersions, especially isotonic aqueoussolutions, dispersions or suspensions which, for example in the case oflyophilised preparations which contain the active ingredient on its ownor together with a carrier can be made up before use. The pharmaceuticalpreparations may be sterilised and/or may contain excipients, forexample preservatives, stabilisers, wetting agents and/or emulsifiers,solubilisers, salts for regulating the osmotic pressure and/or buffersand are prepared in a manner known per se, for example by means ofconventional dissolving or lyophilising processes. The said solutions orsuspensions may contain viscosity-increasing agents or alsosolubilisers.

Suspensions in oil contain as the oil component the vegetable,synthetic, or semi-synthetic oils customary for injection purposes. Inrespect of such, special mention may be made of liquid fatty acid estersthat contain as the acid component a long-chained fatty acid having from8 to 22, carbon atoms. The alcohol component of these fatty acid estershas a maximum of 6 carbon atoms and is a mono- or polyhydric, forexample a mono-, di- or trihydric, alcohol, especially glycol andglycerol.

Pharmaceutical compositions for oral administration can be obtained, forexample, by combining the active ingredient with one or more solidcarriers, if need be granulating a resulting mixture, and processing themixture or granules, if desired, to form tablets or tablet cores, ifneed be by the inclusion of additional excipients.

Orally administrable pharmaceutical compositions also include hardcapsules consisting of gelatin, and also soft, sealed capsulesconsisting of gelatin and a plasticiser, such as glycerol or sorbitol.In soft capsules, the active ingredient is preferably dissolved orsuspended in suitable liquid excipients, to which stabilisers anddetergents may also be added.

Suitable rectally administrable pharmaceutical preparations are, forexample, suppositories that consist of a combination of the activeingredient and a suppository base.

The formulations suitable for parenteral administration are primarilyaqueous solutions ([or example in physiological saline, obtainable bydiluting solutions in polyethylene glycol, of an active ingredient inwater-soluble form, e.g. a water-soluble salt, or aqueous injectablesuspensions containing-viscosity-increasing agents and where appropriatestabilisers. The active ingredient, if need be together with excipients,can also be in the form of a lyophilisate. Solutions such as those used,for example, for parenteral administration can also be employed asinfusion solutions.

The invention relates likewise to a process or a method for thetreatment of one of the pathological conditions mentioned hereinabove,especially a corresponding neoplastic disease. The compounds of formulaI or can be administered as such or especially in the form ofpharmaceutical compositions, prophylactically or therapeutically,preferably in an amount effective against the said diseases, to awarm-blooded animal, for example a human, requiring such treatment. Inthe case of an individual having a bodyweight of about 70 kg the dailydose administered is from approximately 0.001 g to approximately 0.5 g,preferably from approximately 0.005 g to approximately 0.25 g, of acompound of the present invention.

Starting Materials

New starting materials and/or intermediates, as well as processes forthe preparation thereof, are likewise the subject of this invention. Inthe preferred embodiment, such starting materials are used and reactionconditions so selected as to enable the preferred compounds to beobtained.

Starting materials of the formula II and III are known, commerciallyavailable, or can be synthesized in analogy to or according to methodsthat are known in the art.

The aldehyde of formula II, wherein R₁ is hydrogen or lower alkyl whichis unsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino, lower acylamino, R₂ is unsubstituted or substituted heteroaryl, Z is O or a bond,can be obtained, e.g., by a process, wherein an epothilone of formula V

wherein the radicals R₁, R₂ and Z have the meanings as provided for acompound of formula II above, is first reacted with a reagent effectinga retro-aldol reaction furnishing an ester of formula VI

wherein the radicals R₁, R₂ and Z have the meanings as provided for acompound of formula II above, which ester is hydrolized in a second stepinto its components, 4,4-dimethyl-3-hydroxy-5-oxo-heptanoic acid and thealdehyde of formula II as defined above.

Epothilones of formula V which are suitable as starting material for thepreparation of an aldehyde of formula II (see above) are disclosed in J.Org. Chem. 2002, 67, 7730-7736, WO 93/10121, WO97/19086, WO98/38192, WO98108849, WO 98/25929, WO 98/22461, WO99/65013, WO 99/02514, WO99/01124, WO 99/43653, WO99/07692, WO99/67252, WO99/67253, WO00/37473,WO 00/31247 and U.S. Pat. No. 6,194,181 in each case in particular inthe compound claims and the final products of the working examples. Thesubject-matter of the final products of the examples and the claims ishereby incorporated into the present application by reference to thispublications. Comprised are likewise the corresponding stereoisomers aswell as the corresponding crystal modifications, e.g. solvates andpolymorphs, which are disclosed therein.

An ethylketone of formula III,

wherein R₅ is a protecting group and R₃ has the meaning as providedabove for a compound of formula I, can be prepared, e.g., as describedin WO99/07692 on pages 20 to 26 or JP3048641.

An ethylketone of formula III, wherein R₃ is methyl, R₅ is TBDMS and thestereocenter in 4-position having the (S)-configuration, can beprepared, starting with the carboxylic acid ester of formula VII

wherein R₅ represents TBDMS (preparation described by S. Ali and G.Georg in Tetrahedron Letters 38, 10, 1997, 1703-1706, Scheme 2).

In a first step, the carboxylic acid ester of formula VII, wherein R₅represents TBDMS, is transferred into the amide of formula VIII,

wherein R₅ represents TBDMS, by reaction in a suitable solvent liketoluene or benzene with N,O-dimethylhydroxylamine hydrochloride in thepresence of an equal amount of trimethylaluminium at a temperaturebetween −10° C. and +10° C., e.g. about 0° C.

The obtained amide of formula VII, wherein R₅ represents TBDMS, is thenin a second step subject of a Grignard reaction employing ethylmagnesiumbromide or ethyllithium under conditions known as such, e.g. a solutionof the Grignard reagent in diethylether or tetrahydrofuran is dropped tothe solution of the amide of formula VII in the same solvent at atemperature of about 0° C. in which process the reaction mixture can bepunctually warmed or iodine can be added in order to start the reaction.The Grignard reaction is stopped after a period of about 0.5 to 3 hours,e.g. after about 1 hour, providing a ketone of formula IX,

wherein R₅ represents TBDMS.

The obtained ketone of formula IX is then oxidised with the Jonesreagent in a suitable solvent, e.g. acetone, at a temperature betweenabout −5° C. and +5° C., e.g., 0° C., according to a known procedure(see J. Mulzer et al, J. Org. Chem. 1996, 61, 566-572) furnishing thedesired ethylketone of formula III, wherein R₃ is methyl, R₅ is TBDMSand the stereocenter in 4-position is having the (S)-configuration.

Another synthetic route to the intermediate of formula VII starts in afirst step with an antialdol type reaction with anOppolzer-N-propionyl-sultam to give a sultam of formula X

(preparation described by Oppolzer et al, THF 34, 4321 (1993)), whichcan be transformed via Weinreb amidation and subsequent ethyllithium orethylmagnesium bromide addition to an intermediate of formula VII.

Furthermore, the present invention pertains to a method of separatingC4-desmethyl-epothilone B from epothilone G2, which is characterised bychromatography on a Chiralpak-AD column with an eluant containing alower alkane, especially hexane, and a lower alkanol, especially2-propanol.

Additionally, the present invention pertains to a process for theproduction of C4-desmethyl-epothilone B, which comprises the steps of

a) concentrating epothilones in a culture medium for thebiotechnological preparation of epothilones, which medium contains amicroorganism suitable for the preparation of epothilones, water andother suitable customary constituents of culture media, whereby acyclodextrin or a cyclodextrin derivative is added to the medium, or amixture of two or more of these compounds;

b) separating epothilones from one another, which is characterised bychromatography on a reversed-phase column with an eluant containing alower alkylcyanide, wherein chromatography is carried out on columnmaterial charged with hydrocarbon chains, and an eluant containing alower alkylnitrile is used; and wherein, if desired, further working upsteps and purification steps are possible; and

c) finally separating C4-desmethyl-epothilone B from epothilone G2, bychromatography on a Chiralpak-AD column with an eluant containing alower alkane, especially hexane, and a lower alkanol, especially2-propanol.

EXAMPLES

The following Examples serve to illustrate the invention withoutlimiting the invention in its scope. Temperatures are measured indegrees Celsius (° C.). Unless otherwise indicated, the reactions takeplace at room temperature.

Abbreviations

d days

DMSO dimethylsulfoxide

EA ethyl acetate

SC flash chromatography

Me methyl

MS mass spectrometry

rpm rotations per minute

RT room temperature

TBDMS tert-butyl-dimethylsilyl

THF tetrahydrofuran

v volume

General Methods: For flash chromatography, Kieselgel 60 (40-63 μm), andfor thin layer chromatography, DC 60 F₂₅₄-plates from E. Merck(Darmstadt, Germany) are used. All reagents are purchased from Fluka(Buchs, Switzerland).

Example 1 Preparation of C4-Desmethyl-Epothilone B by Fermentation

The volume of harvest from the 500 litre main culture desribed inExample 2D of WO99/42602 of 450 litres is separated into the liquidphase (centrifugate+rinsing water=650 litres) and solid phase (cells=ca.15 kg) using a Westfalia clarifying separator Type SA-20-06 (rpm=6500).The main part of the epothilones are found in the centrifugate, Thecentrifuged cell pulp contains <15% of the determined epothilone portionand is not further processed. The 650 litre centrifugate is then placedin a 4000 litre stirring vessel, mixed with 10 litres of AmberliteXAD-16 (centrifugate:resin volume=65:1) and stirred. After a period ofcontact of ca. 2 h, the resin is centrifuged away in a Heine overflowcentrifuge (basket content 40 litres; rpm=2800). The resin is dischargedfrom the centrifuge and washed with 10-15 litres of deionised water.Desorption of 591.7 kg of charged resin (styrene/divinyl-benzenecopolymer resin XAD-16 charged with epothilones from a culture medium)is effected by stirring the resin in two portions each with 720 litresof toluene in four portions for about 8 hours. Separation of the toluenephase from the resin takes place using a suction filter. The combinedtoluene phases are washed in two portions with each 250 l of water.After phase separation, the toluene extract is concentrated in a 1000litres reactor to approximately 20-40 litres and afterwards concentratedto dryness in a rotary evaporator under vacuum. The toluene extract isdissolved in 16.5 litres of methanol and 24.5 litres of cyclohexane.After addition of 0.8 litres of water phase separation occursimmediately. The methanol fraction is evaporated to dryness in a rotaryevaporator under vacuum. The methanol extract is afterwards beingcrystallized in a solvent mixture consisting of 2.05 litres isopropanoland 10.25 litres cyclohexane, yielding 0.4 kg crystallized material. Thecrystals are dissolved in 3.2 litres acetonitrile/water=⅔ (v/v) and theresulting feed solution is transferred in three separate runs onto apreparative reversed phase column (25 kg RP-18 spherical silica gel,YMC-Gel ODS-A 120; 5-15 μm; Waters Corp., Milford, Mass., USA). Elutionis effected with acetonitrile/water=⅔ (v/v) as mobile phase with a flowrate of 2.7 litres/min; retention time of C4-desmethyl-epothilone B andepothilone G2 58-65 min. Fractionation is monitored with a UV detectorat 250 nm. The acetonitrile of the combined fractions (of the threeruns) having a retention time between 58-65 min is distilled offproviding a mixture of C4-desmethyl-epothilone B and epothilone G₂. 3 gof such mixture is separated into its components on a preparative column(40 cm×10 cm I.D.) containing 2.0 kg amylosetris-(3,5-dimethylphenylcarbamate coated on silicagel (Chiralpak-AD®) inthree runs (1 g each run; mixture being disolved in 20 ml hexane and 20ml 2-propanol). Elution is effected with a hexane/2-propanol 9/1 (v/v)mobile phase with a flow rate of 400 ml/min at room temperature.UV-detection occurs at 249 nm. C4-desmethyl-epothilone B elutes between90 and 110 min. The corresponding fractions are combined and evaporatedto dryness at 40° C. under vacuum and the obtained evaporation residueis re-chromatographed 2 times under the same conditions yielding atleast 71 mg C4-desmethyl-epothilone B with a purity of >97%; ¹H-NMR (500MHz, DMSO-d₆; δ/ppm) 7.31 (s, 1H, H19), 6.51 (s, 1H, H17), 5.26 (d, 9.5Hz, 1H, H15), 5.02 (d, 4.8 Hz, 1H, 3-OH), 4.42 (d, 6.6 Hz, 1H, 7-OH),4.31 (m, 1H, H3), 3.47 (dd, 9.7 Hz, 6.8 Hz, 1H, H7), 3.14 (m, 1H, H4),2.89 (m, 1H, H6), 2.84 (dd, 9.9 Hz, 3.3 Hz, 1H, H13), 2.63 (s, 3H,21-Me), 2.25 (dd, 14.9 Hz, 10.5 Hz, 1H, H2), 2.10 (dd, 14.9 Hz, 2.6 Hz,1H, H2), 2.08 (s, 3H, 16-Me), 2.05 (m, 1H, H14), 1.76 (m, (m, 1H, H14),1.5 (m, 1H, H11), 1.40 (m, 1H, H10), 1.34 (m, 1H, H11), 1.32 (m, 1H,H9), 1.29 (m, 1H, H8), 1.16 (s, 3H, 12-Me), 1.13 (m, 1H, H10), 1.11 (d,7.0 Hz, 3H, 6-Me), 1.05 (m, 1H, H9), 0.93 (d, 6.60 Hz, 3H, 8-Me), 0.89(d, 7.0 Hz, 3H, 4-Me); ESI+MS: [M+H]⁺: 494 D; [M+Na]⁺: 516 D.

Example 2(2S,6R,7S,9S)-6,7-Epoxy-9-hydroxy-2,6,10-trimethyl-11-(2-methyl-4-thiazolyl)-undec-10-en-1-al

300 mg (0.6 mmol) of the compound from stage 2.1 and 0.5 g of hog liveresterase immobilised on Eupergit C (Fluka; 839 U/g) are suspended in 200mL of 1N phosphate buffer (pH=7) and stirred for 3 d. The product isextracted with ethyl acetate and purified by means of FC (150 g ofsilica gel, CH₂Cl₂→CH₂Cl₂/acetone=4:1) giving the desired aldehyde as acolorless oil: R_(f) (CH₂Cl₂/acetone=85:15): 0.36; M+H=338; ¹H-NMR (500MHz, DMSO-d₆; δ/ppm): 9.55 (d, 1.5 Hz, 1H, CHO), 7.29 (s, 1H, H19), 6.44(1s, 1H, H17), 5.1 (d, 5.5 Hz, 1H, 15-OH), 4.10 (m, 1H, H15), 2.81 (m,1H, H13), 2.63 (s, 3H20-Me), 2.36 (m, 1H, H8), 1.18 (s, 3H, 12-Me), 0.99(d, 6Hz, 3H, H9).

Stage 2.1: (3S)-4,4-Dimethyl-3-hydroxy-5-oxo-heptanoic acid((2S,6R,7S,9S)-6,7-epoxy-2,6,10-trimethyl-11-(2-methyl-4-thiazolyl)-undec-10-en-1-al-9-yl)ester

0.5 g (0.99 mmol) of epothilone B are dissolved in 84 mL of CH₂Cl₂.After adding 43 μL (0.44 mmol) of piperidine and 127 μL (0.44 mmol) oftitanium tetra-isopropylate, the reaction solution is strirred for 16 hat RT. After concentration In vacuo, the product is purified by FC (150g of silica gel, CH₂Cl₂→CH₂Cl₂/acetone=4:1) giving a colorless oil:R_(f) (CH₂Cl₂/acetone=85:15): 0.57; M+H=508; ¹H-NMR (500 MHz, DMSO-d₆;δ/ppm): 9.56 (d, 1.5 Hz, 1H CHO), 7.37 (s, 1H, H19), 6.45 (s, 1H, H17),5.31 (m, 1H, H15), 5.10 (d, 5.5 Hz, 1H, 3-OH), 4.13 (m, 1H, H3), 2.74(m, 1H, H13), 2.64 (s, 3H, 20-Me), 2.53 (t, 7.5 Hz, 2H, H6), 2.36 (m,1H, H8), 2.4/2.21 (m/m, 2H, H2), 2.06 (s, 3H, 16-Me), 1.98/1.77 (m/m,2H, H14), 1.67/1.34 (m/m, 2H, H9), 1.45 (m, 2H, H10), 1.18 (s, 3H,4-Me), 1 (s/s/s, 9H, 4-Me, 8-Me, 12-Me), 0.87 (t, 7.5 Hz, 3H, 6-Me).

Example 3(2S,6R,7S,9S)-6,7-Epoxy-9-hydroxy-2,10-dimethyl-11-(2-methyl-4-thiazolyl)-undec-10-en-1-al

550 mg (1.12 mmol) of the compound from stage 3.1 is dissolved in amixture of 200 mL of acetonitrile and 2 mL of 2N NaOH and stirred for 3d. The product is extracted with ethyl acetate and purified by FC (150 gof silica gel, CH₂Cl₂→CH₂Cl₂/acetone=4:1) giving the desired aldehyde acolorless oil: R_(f) (CH₂Cl₂/acetone=85:15): 0.27; M+H=324; ¹H-NMR (500MHz, DMSO-d₆; δ/ppm): 9.56 (d, 1.5 Hz, 1H, CHO), 7.29 (1s, 1H, H19),6.45 (s, 1H, H17), 5.11 (d, 5.5Hz, 15-OH), 4.13 (m, 1H, H15), 3.0 (m,1H, H13), 2.87 (s, 1H, H12), 2.63 (s, 3H, 20-Me), 2.36 (m, 1H, H8), 1.69(m, 2H, H14), 0.99 (d, 6 Hz, 3H, H9).

Stage 3.1: (3S)-4,4-Dimethyl-3-hydroxy-5-oxo-heptanoic acid((2S,6R,7S,9S)-6,7-epoxy-2,10-dimethyl-11-(2-methyl4-thiazolyl)-undec-10-en-1-al-9-yl)ester

1 g (2.03 mmol) of epothilone A is dissolved in 168 mL of CH₂Cl₂. Afteradding 86 μl (0.88 mmol) of piperidine and 254 μl (0.88 mmol) oftitanium tetra-isopropylate, the reaction solution is strirred for 16 hat RT. After concentration in vacuo, the product is purified by FC (200g of silica gel, CH₂Cl₂→CH₂Cl₂/acetone=4:1) giving a colorless oil:R_(f) (CH₂Cl₂/acetone=85:15): 0.55; M+H=494.

Example 4

The following aldehydes of formula II, wherein R₄ is H, can be preparedusing the procedure described in Examples 2 and 3 and using instead ofepothilone A or B as the starting material a compound of formula Vlisted in Table 1. TABLE 1

Aldehyde of formula II, R₄ = H Ex. R₁ R₂ Z R₁ R₂ Z 4.1 Me2-ethyl-4-thiazolyl O Me 2-ethyl-4-thiazolyl O 4.2 Me2-methylthio-4-thiazolyl O Me 2-methylthio-4-thiazolyl O 4.3 Me2-methyl-4-oxazolyl O Me 2-methyl-4-oxazolyl O 4.4 Me2-methyl-benzothiazol- O Me 2-methyl-benzothiazol- O 5-yl 5-yl 4.5 Me2-methyl-benzothiazol- O Me 2-methyl-benzothiazol- O 6-yl 6-yl 4.6 Me5-methyl-2-pyridinyl O Me 5-methyl-2-pyridinyl O 4.7 Me 2-aminomethyt-4-O Me 2-aminomethyl-4- O thiazolyl thiazolyl 4.8 Me 2-dimethylamino-4- OMe 2-dimethylamino-4- O thiazolyl thiazolyl 4.9 Me 2-fluoromethyl-4- OMe 2-fluoromethyl-4- O thiazolyl thiazolyl 4.10 Me 2-methyl-4-thiazolylbond Me 2-methyl-4-thiazolyl bond 4.11 H 2-ethyl-4-thiazolyl O H2-ethyl-4-thiazolyl O 4.12 H 2-methylthio-4-thiazolyl O H2-methylthio-4-thiazolyl O 4.13 H 2-methyl-4-oxazolyl O H2-methyl-4-oxazolyl O 4.14 H 2-methyl-benzothlazol- O H2-methyl-benzothiazol- O 5-yl 5-yl 4.15 Me 2-methyl-benzothiazol- O Me2-methyl-benzothiazol- O 6-yl 6-yl 4.16 H 5-methyl-2-pyridinyl O H5-methyl-2-pyridinyl O 4.17 H 2-aminomethyl-4- O H 2-aminomethyl-4- Othiazolyl thiazolyl 4.18 H 2-dimethylamino-4- O H 2-dimethylamino-4- Othiazolyl thiazolyl 4.19 H 2-fluoromethyl-4- O H 2-fluoromethyl4- Othiazolyl thiazolyl 4.20 H 2-methyl-4-thiazolyl bond H2-methyl-4-thiazolyl O 4.21 Me 2-methyl-benzothiazol- bond Me2-methyl-benzothiazol- bond 5-yl 5-yl

Example 5 TBDMS-ether

Aldehydes of formula II wherein R₄ is TBS can be obtained in accordancewith the procedure described in Example 1c of WO 00/37473 using thealdehydes of formula II from Example 4 as starting materials. Aldehydeof formula II, R₄ = H, from Examples 4.1 to 4.21, 2 and 3 Aldehyde offormula II, R₄ = TBS Ex. R₁ R₂ Z R₁ R₂ Z 5.1 Me 2-ethyl-4-thiazolyl O Me2-ethyl-4-thiazolyl O 5.2 Me 2-methylthio-4-thiazolyl O Me2-methylthio-4-thiazolyl O 5.3 Me 2-methyl-4-oxazolyl O Me2-methyl-4-oxazolyl O 5.4 Me 2-methyl-benzothiazol-5-yl O Me2-methyl-benzothiazol-5-yl O 5.5 Me 2-methyl-benzothiazol- O Me2-methyl-benzothiazol- O 6-yl 6-yl 5.6 Me 5-methyl-2-pyridinyl O Me5-methyl-2-pyridinyl O 5.7 Me 2-aminomethyl-4- O Me 2-aminomethyl-4- Othiazolyl thiazolyl 5.8 Me 2-dimethylamino-4- O Me 2-dimethylamino-4- Othiazolyl thiazolyl 5.9 Me 2-fluoromethyl-4- O Me 2-fluoromethyl-4- Othiazolyl thiazolyl 5.10 Me 2-methyl-4-thiazolyl bond Me2-methyl-4-thiazolyl bond 5.11 H 2-ethyl-4-thiazolyl O H2-ethyl-4-thiazolyl O 5.12 H 2-methylthio-4-thiazolyl O H2-methylthio-4-thiazolyl O 5.13 H 2-methyl-4-oxazolyl O H2-methyl-4-oxazolyl O 5.14 H 2-methyl-benzothiazol- O H2-methyl-benzothiazol- O 5-yl 5-yl 5.15 Me 2-methyl-benzothiazol- O Me2-methyl-benzothiazol- O 6-yl 6-yl 5.16 H 5-methyl-2-pyridinyl O H5-methyl-2-pyridinyl O 5.17 H 2-aminomethyl-4- O H 2-aminomethyl-4- Othiazolyl thiazolyl 5.18 H 2-dimethylamino-4- O H 2-dimethylamino-4- Othiazolyl thiazolyl 5.19 H 2-fluoromethyl-4- O H 2-fluoromethyl-4- Othiazolyl thiazolyl 5.20 H 2-methyl-4-thiazolyl bond H2-methyl-4-thiazolyl bond 5.21 Me 2-methyl-benzothiazol- bond Me2-methyl-benzothiazol- bond 5-yl 5-yl 5.22 Me 2-methyl-4-thiazolyl O Me2-methyl-4-thiazolyl O (from Example 2) 5.23 H 2-methyl-4-thiazolyl O H2-methyl-4-thiazolyl O (from Example 3)

Example 6 (3S,4S)-3-Tert-butyl-dimethylsilyloxy-4-methyl-5-oxo-heptanoicacid

The title compound can be obtained by using the procedure described inExample 2.1 with the title compound from Example 1 as the startingmaterial. The free hydroxy group of the product of this first stage canbe transferred into the corresponding TBDMS ether by the reactionprocedure described in Example 5. After reaction with hog liver esteraseimmobilised on Eupergit C (Fluka; 839 U/g) in 1N phosphate buffer(pH=7), the work-up procedure is modified in order to obtain the titlecompound instead of the aldehyde.

Example 74.8-Dihydroxy-5,5,7,9,13-pentamethyl-16-(2-methyl-benzothiazol-5-yl)-oxacyclohexadec-13-en-2,6-dione(Reference Example)

0.175 ml of trifluoroacetic acid are added dropwise over the course of 5minutes at −20° C. to a solution of 0.041 g of the protected lactone ofstage 7.3 in 0.7 ml of CH₂Cl₂, and the solution is subsequently stirredfor 1 h at 0° C. The solution is then concentrated by evaporation, andthe residue obtained is purified by FC in CH₂Cl₂/methanol 100/1→100/2.The title compound is obtained as a colourless resin; ESI-MS: 502(M+H)⁺. ¹H-NMR (CDCl₃, 300 MHz), δ (ppm vs. TMS): 7.99 (s, 1H); 7.80 (d,1H); 7.36 (d, 1H); 5.92 (d,d, 1H); 5.15-5.28 (m, 1H); 4.21 (d,d, 1H);3.75 (t, 1H); 3.1-3.23 (m, 1H); 2.84 (s, 3H); 1.70 (s, ˜3H).[α]_(D)=−77.39° (c=0.115 in CHCl₃).

Stage 7.1: Carboxylic Acid

3.41 ml of a 1.6 M solution of n-butyllithium in THF is added dropwiseat 0° C. over 15 minutes to a solution of 0.771 ml ofN,N-diisopropyl-ethylamine in 6 ml of THF. The solution is stirred for10 minutes at −4° C./−5° C. and then cooled to −78° C. At thistemperature, a solution of 0.660 g of4,4-dimethyl-3-(tert-butyl-dimethylsilyloxy)-5-oxo-heptanoic acid isadded, the solution is then allowed to warm to −40° C. for 15 minutes,and is subsequently cooled again to −78° C. 3 ml of a solution of 0.608g of the aldehyde of Ex. 5.21 in THF are subsequently added and thesolution is stirred for 30 minutes at −78° C. The reaction is stopped byadding 7 ml of saturated aqueous NH₄Cl solution and, after heating toRT, the solution is mixed with 0.513 ml of acetic acid and extractedwith EA. The combined organic extracts are dried over Na₂SO₄, thesolvent evaporated, and the remaining oily residue purified by FC intoluene/EA 1/1. The obtained aldol product is dissolved in 40 ml ofCH₂Cl₂ and the solution mixed with 0.435 ml of 2,6-lutidine. Aftercooling to 0° C., 0.720 ml of TBS triflate are added and the mixture isstirred for 2½ h at 0° C. After adding 8 ml of 20% citric acid, theorganic phase is separated, the aqueous solution back-extracted withCH₂Cl₂, dried over Na₂SO₄ and the solvent evaporated. The remaining oilis taken up in 20 ml of methanol, the solution is mixed with 2.0 g ofK₂CO₃ and 1 ml of water, and the mixture stirred for 90 minutes at RT.Undissolved constituents are filtered off, the pH value of the filtrateis adjusted to 4.5 with Dowex 50W×8 Ion exchanger resin (very acidiccation exchanger with sulphonic acid groups as the active group, matrixof styrene with 8% DVB as crosslinker, Dowex® is a Trademark of DowChemical Co.), the resin is filtered off and the new filtrateconcentrated by evaporation. The residue is partitioned between 20 ml ofCH₂Cl₂ and 20 ml of saturated aqueous NH₄Cl solution, the organic phaseis separated, the aqueous solution back-extracted with CH₂Cl₂ and thecombined organic extracts are dried over Na₂SO₄ and concentrated byevaporation. The oil thus obtained is purified by FC in CH₂Cl₂/MeOH99/1→99/2. The opbtained material undergoes the above-describedsilylation/desilylation sequence a second time. Finally, pure titlecompound is obtained as an oil; ESI-MS: 862,4 (M+H)⁺.

Stage 7.2: Hydroxy Acid

1.85 ml of a 1 M solution of tetrabutylammonium fluoride are added to asolution of 0.265 g of the carboxylic acid of stage 7.1 in 6 ml of THF,and stirred for 6 hours at RT. Subsequently, 8 ml of EA and 7 ml of 20%citric acid are added, the organic phase is separated and the aqueoussolution back-extracted with EA. The oily residue obtained after dryingthe combined organic extracts over Na₂SO₄ and evaporating the solvent ispurified by FC in CH₂Cl₂/methanol 98/2→97/3. 17 is obtained as an oil.ESI-MS: 748.3 (M+H)⁺. ¹H-NMR (CDCl₃, 200 MHz), δ (ppm vs. TMS): 8.23 (d,1H); 7.76 (d, 1H); 7.40 (d,d, 1H); 5.24 (t, 1H); 4.73-4.84 (m, 1H); 4.45(t, 1H); 3.67-3.74 (m, 1H); 3.10-3.22 (m, 1H); 2.82 (s, 3H); 1.75 (s,3H); 1.14 (d, 3H); 1.08 (d, 3H); 0.80-0.95 (m, ca. 24H); 0.10 (s, 3H);0.05 (s, 3H); 0.04 (s, 3H); 0.01 (s, 3H).

Stage 7.3:4,8-Bis-(tert-butyl-dimethylsilyloxy)-5,5,7,9,13-pentamethyl-16-(2-methyl-benzothiazol-5-yl)-oxacyclohexadec-13-en-2,6-dione

0.0866 ml of triethylamine and 0.0677 ml of 2,4,6-trichlorobenzoylchloride (Aldrich, Buchs, Switzerland) are added to a solution of 0.216g of the carboxylic acid of stage 7.1 in 3 ml of THF, which has beencooled to 0° C., and the solution is stirred for 1 h at 0° C. Thesolution is subsequently added dropwise at RT over 5 minutes to asolution of 0.354 g of N,N-dimethylaminopyridine in toluene and stirredfor 15 h at RT. The solid residue obtained after concentrating thesuspension by evaporation at 35° C. is suspended in 30 ml ofhexane/ether 3/2, filtered and the residue of filtration is washedtwice, each time with 15 ml of the same solvent mixture. The combinedfiltrate is evaporated to dryness and the solid residue is purified byFC twice in toluene/acetone 100/1.25→100/5 or 100/1→100/4, providing thetitle compound as a colourless resin; ESI-MS: 730 (M+H)⁺. ¹H-NMR (CDCl₃,200 MHz), δ (ppm vs. TMS): 7.97 (s, 1H); 7.79 (d, 1H); 7.37 (d, 1H);5.59 (d, 1H); 5.25 (t, 1H); 3.93-4.0 (m, 1H); 3.90 (d, 1H); 2.85 (s,3H); 1.71 (s, 3H). [α]_(D)=−60.720 ° (c=0.415 in CHCl₃).

Example 8-30 C4-Desmethyl-epothilones

C4-Desmethyl-epothilones of formula I can be prepared according to theprocedure described in Example 7 using the aldehydes from Example 5 andthe heptanoic acid from Example 6 C4-Desmethyl-epothilone of formula I,wherein A is O, R₃ is lower Aldehyde from alkyl and R₅ and R₆ arehydrogen Example Example R₁ R₂ Z 8 5.1 Me 2-ethyl-4-thiazolyl O 9 5.2 Me2-methylthio-4-thiazolyl O 10 5.3 Me 2-methyl-4-oxazolyl O 11 5.4 Me2-methyl-benzothiazol-5-yl O 12 5.5 Me 2-methyl-benzothiazol-6-yl O 135.6 Me 5-methyl-2-pyridinyl O 14 5.7 Me 2-aminomethyl-4-thiazolyl O 155.8 Me 2-dimethylamino-4-thiazolyl O 16 5.9 Me2-fluoromethyl-4-thiazolyl O 17 5.10 Me 2-methyl-4-thiazolyl bond 185.11 H 2-ethyl-4-thiazolyl O 19 5.12 H 2-methylthio-4-thiazolyl O 205.13 H 2-methyl-4-oxazolyl O 21 5.14 H 2-methyl-benzothiazol-5-yl O 225.15 Me 2-methyl-benzothiazol-6-yl O 23 5.16 H 5-methyl-2-pyridinyl O 245.17 H 2-aminomethyl-4-thiazolyl O 25 5.18 H 2-dimethylamino-4-thiazolylO 26 5.19 H 2-fluoromethyl-4-thiazolyl O 27 5.20 H 2-methyl-4-thiazolylbond 28 5.21 Me 2-methyl-benzothiazol-5-yl bond 29 5.22 Me2-methyl-4-thiazolyl O 30 5.23 H 2-methyl-4-thiazolyl O

Example 31 (Aldolisation Step: Preparation of C4-BisNor-EPO-B)

A solution of in situ TMS-disilylated 3-(R)-hydroxy-5-oxo-heptanoic acid(2 mmol) in 4 ml of THF, obtained from yeast reduction (Mitsubishi KaseiCorp. JP3048641, 1991-03-03), is cooled to −10° C. and treated with 2.2mmol of LDA in THF. The solution is stirred for 20 min. and then cooleddown to −40° C. To this lithium enolate is added a solution of protectedaldehyde of example 5.22 (2.5 mmol) in dry THF.

The reaction mixture is warmed up to −30° C. and kept at thistemperature for 2-3 hours. Finally the reaction mixture is quenched withaqueous citric acid solution. The organic phase separated and theaqueous phase is twice extracted with EtOAc. The combined organic phasesare carefully concentrated in vacuo. The residue is dissolved in EtOAcand washed with water and brine. The EtOAc phase is dried over anhydrousNa₂SO₄ and finally evaporated to dryness to give a viscous oil of thealdol product.

This product is further transformed by selective deprotection at OR₄ andmacrolactoni-sation he desired bis-nor-Epothilone derivative undersimilar conditions as described in Example 7

Example 32 C4-BisNor-epothilones

C4-BisNor-epothilones of formula I can be prepared according to theprocedure described in Example 31 using the protected aldehydes fromExample 5 and the heptanoic acid from Example 31 C4-BisNor-epothilone offormula I, wherein A is O and R₃, Aldehyde from R₅ and R₆ are hydrogenExample Example R₁ R₂ Z 32 5.1 Me 2-ethyl-4-thiazolyl O 33 5.2 Me2-methylthio-4-thiazolyl O 34 5.3 Me 2-methyl-4-oxazolyl O 35 5.4 Me2-methyl-benzothiazol-5-yl O 36 5.5 Me 2-methyl-benzothiazol-6-yl O 375.6 Me 5-methyl-2-pyridinyl O 38 5.7 Me 2-aminomethyl-4-thiazolyl O 395.8 Me 2-dimethylamino-4-thiazolyl O 40 5.9 Me2-fluoromethyl-4-thiazolyl O 41 5.10 Me 2-methyl-4-thiazolyl bond 425.11 H 2-ethyl-4-thiazolyl O 43 5.12 H 2-methylthio-4-thiazolyl O 445.13 H 2-methyl-4-oxazolyl O 45 5.14 H 2-methy-benzothiazol-5-yl O 465.15 Me 2-methyl-benzothiazol-6-yl O 47 5.16 H 5-methyl-2-pyridinyl O 485.17 H 2-aminomethyl-4-thiazolyl O 49 5.18 H 2-dimethylamino-4-thiazolylO 50 5.19 H 2-fluoromethyl-4-thiazolyl O 51 5.20 H 2-methyl-4-thiazolylbond 52 5.21 Me 2-methyl-benzothiazol-5-yl bond 53 5.22 Me2-methyl-4-thiazolyl O 54 5.23 H 2-methyl-4-thiazolyl O

Example 55 Dry Capsules

3000 capsules, each of which contain 0.005 g of one of theC4-desmethyl-epothilone of the formula I mentioned in the precedingExamples as active ingredient, are prepared as follows: CompositionActive ingredient 1.50 g Lactose 750.00 g Avicel PH 102 300.00 g(microcrystalline cellulose) Polyplasdone XL 30.00 g(polyvinylpyrrolidone) Magnesium stearate 9.00 g

Preparation process: The active ingredient is passed through a No. 30hand screen. The active ingredient, lactose, Avicel PH 102 andPolyplasdone XL are blended for 15 minutes in a mixer. The blend isgranulated with sufficient water (about 500 mL), dried in an oven at 35°C. overnight, and passed through a No. 20 screen. Magnesium stearate ispassed through a No. 20 screen, added to the granulation mixture, andthe mixture is blended for 5 minutes in a mixer. The blend isencapsulated in No. 0 hard gelatin capsules each containing an amount ofthe blend equivalent to 25 mg of the active ingredient.

Example 56 PEG Solution

5 mg of a C4-desmethyl-epothilone of formula I is dissolved in 98-100%propylene glycol (1.0 ml). The solution is sterile filtered through a0.22 microns pore size filter and charged to 1 ml ampoules. The filledampoules are used for storage and shipment. Prior to intravenousadministration, the contents of an ampoule are added to 250 to 1000 mlof a 5% glucose solution in water-for-injection.

Example 57

The efficacy of a C4-desmethyl-epothilone of formula I as inhibitor ofmicrotubule depolymerisation can be determined by the test proceduredescribed above. Final assay concentrations of test compound and porcinemicrotubule protein (Batch #9) were 4 uM and 0.8 mg/ml, respectively.TABLE 2 Tubulin Polymerisation Compound tested (% of control) Example 193.3 Epothilone A (Reference Example) 76.5 Epothilone B (ReferenceExample) 93.3 Paclitaxel (Reference Example) 62.1

Example 58

The efficacy against tumour cells can be demonstrated in the proceduredescribed above. TABLE 3 Epothilone B Paclitaxel (Reference (ReferenceCell Growth Inhibition Example 1 Example) Example) KB-31, IC₅₀ (nM) 0.49± 0.05 0.28 ± 0.04 3.76 ± 0.52 KB-8511, IC₅₀ (nM) 0.80 ± 0.16 0.20 ±0.04 739 ± 86 

1. An epothilone of formula I

wherein A represents O or NR₇; R₁ is hydrogen or lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkanoylin free or protected form, lower alkoxy, halogen, amino, lower alkylamino, di-lower alkyl amino, or lower acyl amino; R₂ is unsubstituted orsubstituted heteroaryl having at least one nitrogen atom, R₃ representshydrogen or lower alkyl; R₅ and R₆ are hydrogen, R₇ is hydrogen or loweralkyl; Z is O or a bond; with the proviso that when R₂ is2-methyl-thiazolyl and Z is O, R₁ represents lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino, and when R₂ is 2-methyl-thiazolyl and Z is a bond, R representslower alkyl which is substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, or a salt thereof:
 2. An epothilone of formula Ia or Ib

wherein A represents O or NR₇; R₁ is hydrogen or lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkanoylin free or protected form, lower alkoxy, halogen, amino, lower alkylamino, all-lower alkyl amino, or lower acyl amino; R₂ is unsubstitutedor substituted heteroaryl having at least one nitrogen atom, R₃represents hydrogen or lower alkyl; R₅ and R₆ are hydrogen, and R₇ ishydrogen or lower alkyl; Z is O or a bond; under the proviso that whenR₂ is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino; and when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ representslower alkyl which is substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino; or a salt thereof.
 3. The epothilone of formula I accordingto claim 1, wherein A represents O or NR₇; R₁ is hydrogen or lower alkydwhich is unsubstituted or substituted by hydroxy, lower acyloxy, loweralkanoyl in free or protected form, lower alkoxy, halogen, amino, loweralkyl amino, di-lower alkyl amino, or lower acyl amino; R₂ is thiazolyl,oxazolyl, pyridyl, benzothiazolyl, benzoxazolyl or benzoimidazolyl,which in each case is substituted or unsubstituted; R₃ representshydrogen or lower alkyl; R₅ and R₆ are hydrogen; R₇ is hydrogen or loweralkyl; Z is O or a bond; with the proviso that when R₂ is2-methyl-thiazolyl and Z is O, R₁ represents lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino; and when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ representslower alkyl which is substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, or a salt thereof.
 4. The epothilone of formula I accordingto claim 1, wherein A represents O or NR₇; R₁ is hydrogen or lower alkylwhich is unsubstituted or substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino; R₂ is thiazolyl, oxazolyl, pyridyl, benzothiazolyl, which ineach case is substituted or unsubstituted; R₃ represents hydrogen orlower alkyl; R₅ and R₆ are hydrogen; R₇ is hydrogen or lower alkyl; Z isO or a bond with the proviso that when R₂ is 2-methyl-thiazolyl and Z isO, R₁ represents lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino,di-lower alkyl amino or lower acyl amino; and when R₂ is2-methyl-thiazolyl and Z is a bond, R₁ represents lower alkyl which issubstituted by hydroxy, lower acyloxy, lower alkoxy, halogen, amino,lower alkyl amino, di-lower alkyl amino or lower acyl amino or a saltthereof.
 5. The epothilone of formula I according to claim 1, wherein Arepresents O; R₁ is hydrogen or lower alkyl; R₂ is 2-methyl-thiazolyl,2-ethyl-thiazolyl, 2-methylthio-thiazolyl, 2-aminomethyl-thiazolyl,2-dimethylamino-thiazolyl, 2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl,3-methyl-pyridinyl, 2-methyl-benzothiazolyl; R₃ represents hydrogen orlower alkyl; R₅ and R₆ are hydrogen; Z is O or a bond; with the provisothat when R₂ is 2-methyl-thiazolyl, Z is O and R₁ represents loweralkyl, or a salt thereof.
 6. The epothilone of formula I according toclaim 1, wherein A represents O; R₁ is hydrogen or lower alkyl; R₂ is2-methyl-thiazolyl, 2-ethyl-thiazolyl, 2-methylthio-thiazolyl,2-aminomethyl-thiazolyl, 2-dimethylamino-thiazolyl,2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl, 3-methyl-pyridinyl,2-methyl-benzothiazolyl; R₃ represents methyl; R₅ and R₆ are hydrogen; Zis O or a bond; with the proviso that when R₂ is 2-methyl-thiazolyl, Zis O and R₁ represents lower alkyl, or a salt thereof.
 7. Apharmaceutical composition, comprising an epothilone of formula Iaccording to claim 1, or a pharmaceutically acceptable salt thereof,provided that salt-forming groups are present, and one or morepharmaceutically acceptable carriers. 8-9. (canceled)
 10. A method fortreatment of warm-blooded animals, including humans, in which antherapeutically effective amount of an epothilone of the formula Iaccording to claim 1 or a pharmaceutically acceptable salt of such acompound is administered to a warm-blooded animal suffering from atumour disease.
 11. A process for the preparation of an epothilone offormula I,

wherein A represents O or NR₇; R₁ is hydrogen or lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino; R₂ is unsubstituted or substituted heteroaryl having at least onenitrogen atom; R₃ represents hydrogen or lower alkyl; R₅ and R₆ arehydrogen; and R₇ is hydrogen or lower alkyl; and Z is O or a bond; or apharmaceutically acceptable salt thereof: comprising the steps of: (a)reacting an aldehyde of formula II

wherein R₁, R₂ and Z have the meanings as provided above for a compoundof formula I and R₄ is a protecting group, with an ethylketone offormula III,

wherein R₅ is H or a protecting group different or identical to R₄ andR₃ has the meaning as provided above for a compound of formula I, toprovide the aldol of formula IV,

wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, R₄ a protecting group, R₅ is H or a protectinggroup different or identical to R₄ and R₆ is hydrogen (b) reacting thealdol of formula IV with a reagent capable to introduce a protectinggroup which is different or identical to R₄ furnishing a carboxylic acidof formula IV, wherein R₁, R₂, R₃ and Z have the meanings as providedabove for a compound of formula I, R₄ a protecting group and R₅ is H orR₅ and R₆ are protecting groups different or identical to R₄; (c)reacting the carboxylic acid of formula IV with a reagent capable toremove the protecting group R₄ under conditions which do not result inthe removal of the protecting groups R₅ and R₆ providing a carboxylicacid of formula IV, wherein R₁, R₂, R₃ and Z have the meanings asprovided above for a compound of formula I, R₄ is hydrogen and R₅ is Hor R₅ is H or R₅ and R₆ are protecting groups, (d) macrolactonizing thecarboxylic acid of formula IV providing the epothilone of formula I,wherein R₁, R₂, R₃ and Z have the meanings as provided above for acompound of formula I, A is O and R₅ is H or R₅ and R₆ are protectinggroups; (e) reacting the epothilone of formula I with a reagent capableof removing the protecting groups R₅ and R₆ furnishing an epothilone offormula I, wherein R₁, R₂, R₃, R₅, R₆ and Z have the meanings asprovided above for a compound of formula I and A is O; and (f)optionally reacting the epothilone of formula I into an epothilone offormula I wherein R₁, R₂, R₃, R₅, R₆ and Z have the meanings as providedabove for a compound of formula I and A is NR₇, wherein R₇ is hydrogenor lower alkyl.
 12. An ethylketone of formula III,

wherein R₃ has the meaning as provided above for a compound of formula Iand R₅ is hydrogen or a protecting group.
 13. An aldol of formula IV,

R₁ is hydrogen or lower alkyl which is unsubstituted or substituted byhydroxy, lower acyloxy, lower alkoxy, halogen, amino, lower alkyl amino,di-lower alkyl amino, lower acyl amino, R₂ is unsubstituted orsubstituted heteroaryl; R₃ represents hydrogen or lower alkyl; R₄ ishydrogen or a protecting group; R₅ is a protecting group different oridentical to R₄; R₆ is hydrogen or a protecting group different oridentical to R₄; and Z is O or a bond.
 14. A process for the preparationof an aldehyde of formula II

wherein R₁ is hydrogen or lower alkyl which is unsubstituted orsubstituted by hydroxy, lower acyloxy, lower alkoxy, halogen, amino,lower alkyl amino, di-lower alkyl amino, lower acyl amino; R₂ isunsubstituted or substituted heteroaryl; Z is O or a bond; comprisingthe steps of: (a) reacting an epothilone of formula V

wherein the radicals R₁, R₂ and Z have the meanings as provided for acompound of formula II above, with a reagent effecting a retro-aldolreaction furnishing an ester of formula VI

wherein the radicals R₁, R₂ and Z have the meanings as provided for acompound of formula II above, which ester is hydrolized in a second stepinto its components, 4,4-dimethyl-3-hydroxy-5oxo-heptanoic acid and thealdehyde of formula II as defined above.
 15. A method of separatingC4-desmethyl-epothilone B from epothilone G2, which is characterized bychromatography on a Chiralpak-AD column with an eluant containing alower alkane and a lower alkanol.
 16. A process for the production ofC4-desmethyl-epothilone B, which comprises the steps of a) concentratingepothilones in a culture medium for the biotechnological preparation ofepothilones, which medium contains a microorganism suitable for thepreparation of epothilones, water and other suitable customaryconstituents of culture media, whereby a cyclodextrin or a cyclodextrinderivative is added to the medium, or a mixture of two or more of thesecompounds; b) separating epothilones from one another, which ischaracterized by chromatography on a reversed-phase column with aneluant containing a lower alkylcyanide, wherein chromatography iscarried out on column material charged with hydrocarbon chains, and aneluant containing a lower alkyinitrile is used; and wherein, if desired,further working up steps and purification steps are possible; and c)finally separating C4-desmethyl-epothilone B from epothilone G2, bychromatography on a Chiralpak-AD column with an eluant containing alower alkane, and a lower alkanol.
 17. The epothilone of formula Ia orIb according to claim 2, wherein A represents O or NR₇; R₁ is hydrogenor lower alkyd which is unsubstituted or substituted by hydroxy, loweracyloxy, lower alkanoyl in free or protected form, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino, or lower acylamino; R₂ is thiazolyl, oxazolyl, pyridyl, benzothiazolyl, benzoxazolylor benzoimidazolyl, which in each case is substituted or unsubstituted;R₃ represents hydrogen or lower alkyl; R₅ and R₆ are hydrogen; R₇ ishydrogen or lower alkyl; Z is O or a bond; with the proviso that when R₂is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino; and when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ representslower alkyl which is substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino, or a salt thereof.
 18. The epothilone of formula Ia or Ibaccording to claim 2, wherein A represents O or NR₇; R₁ is hydrogen orlower alkyl which is unsubstituted or substituted by hydroxy, loweracyloxy, lower alkoxy, halogen, amino, lower alkyl amino, di-lower alkylamino or lower acyl amino; R₂ is thiazolyl, oxazolyl, pyridyl,benzothiazolyl, which in each case is substituted or unsubstituted; R₃represents hydrogen or lower alkyl; R₅ and R₆ are hydrogen; R₇ ishydrogen or lower alkyl; Z is O or a bond; with the proviso that when R₂is 2-methyl-thiazolyl and Z is O, R₁ represents lower alkyl which isunsubstituted or substituted by hydroxy, lower acyloxy, lower alkoxy,halogen, amino, lower alkyl amino, di-lower alkyl amino or lower acylamino; and when R₂ is 2-methyl-thiazolyl and Z is a bond, R₁ representslower alkyl which is substituted by hydroxy, lower acyloxy, loweralkoxy, halogen, amino, lower alkyl amino, di-lower alkyl amino or loweracyl amino or a salt thereof.
 19. The epothilone of formula Ia or Ibaccording to claim 2, wherein A represents O; R₁ is hydrogen or loweralkyl; R₂ is 2-methyl-thiazolyl, 2-ethyl-thiazolyl,2-methylthio-thiazolyl, 2-aminomethyl-thiazolyl,2-dimethylamino-thiazolyl, 2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl,3-methyl-pyridinyl, 2-methyl-benzothiazolyl; R₃ represents hydrogen orlower alkyl; R₅ and R₆ are hydrogen; Z is O or a bond; with the provisothat when R₂ is 2-methyl-thiazolyl, Z is O and R₁ represents loweralkyl, or a salt thereof.
 20. The epothilone of formula Ia or Ibaccording to claim 2, wherein A represents O; R₁ is hydrogen or loweralkyl; R₂ is 2-methyl-thiazolyl, 2-ethyl-thiazolyl,2-methylthio-thiazolyl, 2-aminomethyl-thiazolyl,2-dimethylamino-thiazolyl, 2-fluoromethyl-thiazolyl, 2-methyl-oxazolyl,3-methyl-pyridinyl, 2-methyl-benzothiazolyl; R₃ represents methyl; R₅and R₆ are hydrogen; Z is O or a bond; with the proviso that when R₂ is2-methyl-thiazolyl, Z is O and R₁ represents lower alkyl, or a saltthereof.
 21. A pharmaceutical composition, comprising an epothilone offormula Ia or Ib according to claim 2, or a pharmaceutically acceptablesalt thereof, provided that salt-forming groups are present, one or morepharmaceutically acceptable carriers.
 22. A method for treatment ofwarm-blooded animals, including humans, in which an therapeuticallyeffective amount of an epothilone of the formula Ia or Ib according toclaim 2 or a pharmaceutically acceptable salt of such a compound isadministered to a warm-blooded animal suffering from a tumour disease.